Conventionally, in JP-A-7-75544 and JP-A-7-303469, polymerase chain reaction (PCR) is reported as a method of synthesizing a nucleotide sequence from an existing sequence.
The polymerase chain reaction (PCR) is a method in which a target DNA is flanked by a pair of primers, a DNA polymerase is allowed to act on it repeatedly, whereby the region flanked by the primers can be continuously amplified.
By PCR, only the target sequence can be substantially correctly amplified to produce a large number of copies, further an efficient amplification is possible in a short time, therefore, PCR is widely used for a variety of studies, assays, tests and the like in biochemical, medical fields, etc., at present.
It has been conventionally assumed that the principle of PCR is to control temperature, and the reaction is performed by repeating heating and cooling procedures (thermal cycle). More specifically, for example, by denaturing a double-stranded DNA molecule, which is the target for amplification, into complementary single strands at a high temperature, annealing a primer, which has been selected to be complementary to a part of the DNA, to the strand by cooling, and extending the DNA to the downstream of the primer with a DNA polymerase by heating again, and so on, one cycle consisting of the steps of denaturing, annealing and extending is repeated multiple times, whereby a large amount of double-stranded DNA can be amplified.
Specifically, it is necessary to repeat the thermal cycle, which consists of 1) raising the temperature of a sample to 95° C. in order to disrupt the hydrogen bond of the double-stranded DNA, 2) subsequently, lowering the temperature of the sample to 45° C. in order to recombine the DNA to a primer for replication, 3) further, raising the temperature of the sample to 74° C. in order to replicate the DNA by extending the primer with a heat-resistant polymerase, a number of times. In such a DNA amplification reaction, the foregoing thermal cycle was carried out by putting a sample into a container made of synthetic resin or the like, and accommodating this container into an aluminum block.
However, the foregoing thermal cycle consumed a lot of time, and it took several hours to obtain a required amount of DNA. In addition, when reaction is carried out by controlling the temperature (heating and cooling), there is a limit on changing the temperature in an instant, and changeover of each step cannot be performed smoothly, whereby the accuracy of the nucleotide sequence to be amplified may be affected, or DNA other than the target may be replicated in some cases. Further, in order to change the temperature rapidly, a special apparatus or technique is needed, therefore there are an economic problem such as investment in equipment and a technical problem.
In view of the foregoing problems, as a support capable of easily immobilizing DNA and suitable for replicating DNA by DNA amplification reaction, in WO 00/22108, WO 02/12891 or JP-A-2002-82116, a solid support which comprises, on the surface of a substrate, a surface-treated layer and chemically modified layer having a functional group capable of covalently binding to a nucleic acid molecule sequentially has been developed.
However, the amount of DNA immobilized on the foregoing solid support and the bond strength to DNA are not always sufficient, therefore, the emergence of a solid support capable of immobilizing DNA in a higher proportion and with a higher bond strength to DNA has been awaited.
The object of the present invention is to provide a solid support capable of immobilizing nucleic acid molecules in a higher proportion, and with a higher bond strength to nucleic acid molecules.